Investigations into novel signalling pathways in the response to viral and bacterial infections

Our current understanding of the host response to pathogenic insult is in constant flux. The long term goal is to better understand the host response so as to design better therapeutics with higher efficacies and less side effects. Thus, the current work sought to increase our understanding the immune response to viral and bacterial perturbation of host signalling pathways. The TIR-domain containing adaptor TRAM is a relatively understudied protein with regard to TLR signalling, particularly with respect to type-I inferferon production. A re-evaluation of the role of TRAM in TLR7 signalling showed that TRAM is required for maximal levels of TLR7 mediated RANTES, CXCL10 and IFNβ cytokine secretion but not TNFα production. TLR7 signalling was shown to activate IRF3 and NFκB in murine bone marrow derived macrophages. However, while TLR7 mediated IRF3 activation was TRAM dependent, NFκB activation was not. TRAM was also shown to mediate signalling via a physiological activator of TLR7, human rhinovirus 16 (HRV16). TRAM’s role in TLR7 is hypothesised to be dependent on its ability to membrane localise as overexpression of a TRAM myriostoylation mutant which is incapable of membrane localisation dose dependently inhibited TLR7 mediated activation of RANTES, IFNβ, IFNα but not NFκB reporter genes. Furthermore, TRAM was shown to co-immunoprecipitate with the TLR7 adaptor molecule MyD88 upon TLR7 activation. This is first time that either IRF3 or TRAM has been shown to play role in TLR7 signalling. The second part of this project focused on the characterisation of the proteomic response to two respiratory pathogens, HRV16 and Bordetella pertussis (B. pertussis) in a lung epithelial cell line using mass spectrometry. Significant alternations were observed in the host proteome in response to both pathogens with proteins involved in the immune response, redox signalling, cancer related pathways, metabolism and DNA binding being particularly well represented. There was also a significant overlap between proteins identified in response to both infections with immune response proteins being responsible for a third of the overlap. Supression of endogenous levels of a number of protein hits prior to infection with either HRV16 or B. pertussis did not affect levels of cytokine secretion. However, suppression of two proteins, the microtubule regulator stathmin 1 and the protein phosphatase PPP1Cα led to a significant decrease in TLR4 mediated IL-6 production. Thus the current work has indicated novel roles for a number of proteins in the host response to pathogen challenge. TRAM is required for maximal TLR7 mediated anti-viral cytokine secretion and both stathmin 1 and PPP1Cα are required for maximal TLR4 mediated IL-6 production